scholarly journals The CSC connects three major axonemal complexes involved in dynein regulation

2012 ◽  
Vol 23 (16) ◽  
pp. 3143-3155 ◽  
Author(s):  
Thomas Heuser ◽  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Elizabeth F. Smith ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Structural Heterogeneity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Localization Model ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
Health And Development ◽  
Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

scholarly journals DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

2015 ◽  
Vol 26 (15) ◽  
pp. 2788-2800 ◽  
Author(s):  
Junya Awata ◽  
Kangkang Song ◽  
Jianfeng Lin ◽  
Stephen M. King ◽  
Michael J. Sanderson ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Motor Domain ◽  
Flagellar Motility ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
And Function ◽  
Linker Domain ◽  
Distal Lobe

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

scholarly journals Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin–dynein regulatory complex

2019 ◽  
Vol 116 (46) ◽  
pp. 23152-23162 ◽  
Author(s):  
Long Gui ◽  
Kangkang Song ◽  
Douglas Tritschler ◽  
Raqual Bower ◽  
Si Yan ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Wild Type ◽  
Core Complex ◽  
The Core ◽  
C Terminus ◽  
Cryo Electron Tomography ◽  
Regulatory Complex ◽  
Cilia And Flagella ◽  
Linker Domain ◽  
Shed Light

The nexin–dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits,” namely DRC3/5–8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

scholarly journals The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

2006 ◽  
Vol 17 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Chun Yang ◽  
Pinfen Yang
Keyword(s):  
Regulatory Proteins ◽  
Flagellar Motility ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Wide Range ◽  
Anchoring Protein ◽  
Radial Spokes ◽  
Dependent Protein ◽  
Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

scholarly journals Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

2019 ◽  
Author(s):  
Long Gui ◽  
Kangkang Song ◽  
Douglas Tristchler ◽  
Raqual Bower ◽  
Yan Si ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Core Complex ◽  
Ciliary Motility ◽  
C Terminus ◽  
Large N ◽  
Cryo Electron Tomography ◽  
N Terminus ◽  
Regulatory Complex ◽  
Cilia And Flagella ◽  
Linker Domain

ABSTRACTThe nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits” associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.Significance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1–11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

scholarly journals Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

2012 ◽  
Vol 23 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Cynthia F. Barber ◽  
Thomas Heuser ◽  
Blanca I. Carbajal-González ◽  
Vladimir V. Botchkarev ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Flagellar Motility ◽  
Three Dimensional Structure ◽  
Radial Spoke ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Axonemal Dynein ◽  
Subtomogram Averaging

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.

scholarly journals The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility

2013 ◽  
Vol 201 (2) ◽  
pp. 263-278 ◽  
Author(s):  
Ryosuke Yamamoto ◽  
Kangkang Song ◽  
Haru-aki Yanagisawa ◽  
Laura Fox ◽  
Toshiki Yagi ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Beat Frequency ◽  
Coiled Coil ◽  
Chain Complex ◽  
Flagellar Motility ◽  
Flagellar Beat ◽  
Cryo Electron Tomography ◽  
Regulatory Complex ◽  
Physical Interactions ◽  
Dynein Arms

Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin–dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.

scholarly journals Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽  
2021 ◽  
Vol 371 (6525) ◽  
pp. eabd4914
Author(s):  
Sudarshan Gadadhar ◽  
Gonzalo Alvarez Viar ◽  
Jan Niklas Hansen ◽  
An Gong ◽  
Aleksandr Kostarev ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Male Fertility ◽  
Electron Tomography ◽  
Structural Basis ◽  
Cellular Functions ◽  
Flagellar Beat ◽  
Cryo Electron Tomography ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

scholarly journals Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

2011 ◽  
Vol 22 (7) ◽  
pp. 976-987 ◽  
Author(s):  
Yong Yang ◽  
Deborah A. Cochran ◽  
Mary D. Gargano ◽  
Iryna King ◽  
Nayef K. Samhat ◽  
...  
Keyword(s):  
Flagellar Motility ◽  
Genetic Screens ◽  
Sperm Flagellum ◽  
Respiratory Epithelia ◽  
Downstream Effectors ◽  
Radial Spokes ◽  
Flagellar Proteins ◽  
Flagellar Protein ◽  
Cilia And Flagella ◽  
Lost Boys

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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